Bottom Line:
We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains.Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains.Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

ABSTRACTThe ubiquitin receptors Rad23 and Dsk2 deliver polyubiquitylated substrates to the proteasome for destruction. The C-terminal ubiquitin-associated (UBA) domain of Rad23 functions as a cis-acting stabilization signal that protects this protein from proteasomal degradation. Here, we provide evidence that the C-terminal UBA domains guard ubiquitin receptors from destruction by preventing initiation of degradation at the proteasome. We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains. Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains. Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

f4: A conserved motif contributes to the protective effect.(a) Schematic representation of the generation of chimeric UBA1/UBA2 domains. (b) Relative fluorescence levels of yeast cells expressing Ub-R-GFP with C-terminal UBA1, UBA2 and chimeric UBA domains analysed by flow cytometry. Values are means and standard deviations (n=3). *P<0.05, **P<0.01 (Student's t-test). (c) Alignment of the Xaa-Gly-Phe/Tyr-Xaa motif in the loop between helix 1 and helix 2 of a number of UBA domains. (d) Relative fluorescence levels of yeast cells expressing Ub-R-GFP-UBA1, Ub-R-GFP-UBA1Y160F, Ub-R-GFP-UBA2 and Ub-R-GFP-UBAF369Y analysed by flow cytometry (the numbers refer to positions of these amino acids in Rad23). Values are means and standard deviations (n=6). *P<0.05, **P<0.01 (Student's t-test). (e) Turnover of Ub-R-GFP-UBA1 and Ub-R-GFP-UBA1Y160F. Samples were taken at the indicated time points and probed with a GFP-specific antibody.

Mentions:
We were intrigued by the fact that the UBA1 and UBA2 domains displayed such a striking difference in their ability to protect proteins from proteasomal degradation, despite their structural resemblance. Both domains adopt a fold with three tightly packed α-helix bundles in a conformation that is characteristic of UBA domains27. In an attempt to pinpoint the difference between the domains responsible for the divergent effects on proteasomal degradation, we generated chimeric UBA domains in which fragments containing the first, second and third helices from the UBA1 and UBA2 domains were combined (Fig. 4a). We found that most C-terminal helices of the UBA2 domain could be replaced with the equivalent fragment of the UBA1 domain, with only a modest impact on its protective effect, whereas the first and the second helices appeared to be more important for the stabilizing effect (Fig. 4b). Notably, the loop between the first two helices contains a highly conserved motif (Xaa-Gly-Phe/Tyr-Xaa) that is found to be important for specific binding events27 as well as for ubiquitin-binding-assisted conformational switch28. We noticed that all protective UBA domains so far tested harboured a phenylalanine residue in this motif, whereas the UBA1 domains from Rad23 and the human homologue Rad23-A (hHR23A), which are both unprotective17, had a tyrosine residue at the same position (Fig. 4c). Remarkably, we found that replacing the tyrosine residue in this motif in the UBA1 domain with a phenylalanine residue resulted in a UBA1 domain that significantly increased the steady-state levels of the reporter substrate, although not to the same extent as the wild-type UBA2 domain (Fig. 4d). Turnover analysis showed that this single amino-acid substitution in the UBA1 domain indeed prolonged the half-life of the substrate (Fig. 4e). Our data suggest that the protective effect of the UBA2 domain is an intrinsic feature and identifies a well-conserved motif as a contributing factor.

f4: A conserved motif contributes to the protective effect.(a) Schematic representation of the generation of chimeric UBA1/UBA2 domains. (b) Relative fluorescence levels of yeast cells expressing Ub-R-GFP with C-terminal UBA1, UBA2 and chimeric UBA domains analysed by flow cytometry. Values are means and standard deviations (n=3). *P<0.05, **P<0.01 (Student's t-test). (c) Alignment of the Xaa-Gly-Phe/Tyr-Xaa motif in the loop between helix 1 and helix 2 of a number of UBA domains. (d) Relative fluorescence levels of yeast cells expressing Ub-R-GFP-UBA1, Ub-R-GFP-UBA1Y160F, Ub-R-GFP-UBA2 and Ub-R-GFP-UBAF369Y analysed by flow cytometry (the numbers refer to positions of these amino acids in Rad23). Values are means and standard deviations (n=6). *P<0.05, **P<0.01 (Student's t-test). (e) Turnover of Ub-R-GFP-UBA1 and Ub-R-GFP-UBA1Y160F. Samples were taken at the indicated time points and probed with a GFP-specific antibody.

Mentions:
We were intrigued by the fact that the UBA1 and UBA2 domains displayed such a striking difference in their ability to protect proteins from proteasomal degradation, despite their structural resemblance. Both domains adopt a fold with three tightly packed α-helix bundles in a conformation that is characteristic of UBA domains27. In an attempt to pinpoint the difference between the domains responsible for the divergent effects on proteasomal degradation, we generated chimeric UBA domains in which fragments containing the first, second and third helices from the UBA1 and UBA2 domains were combined (Fig. 4a). We found that most C-terminal helices of the UBA2 domain could be replaced with the equivalent fragment of the UBA1 domain, with only a modest impact on its protective effect, whereas the first and the second helices appeared to be more important for the stabilizing effect (Fig. 4b). Notably, the loop between the first two helices contains a highly conserved motif (Xaa-Gly-Phe/Tyr-Xaa) that is found to be important for specific binding events27 as well as for ubiquitin-binding-assisted conformational switch28. We noticed that all protective UBA domains so far tested harboured a phenylalanine residue in this motif, whereas the UBA1 domains from Rad23 and the human homologue Rad23-A (hHR23A), which are both unprotective17, had a tyrosine residue at the same position (Fig. 4c). Remarkably, we found that replacing the tyrosine residue in this motif in the UBA1 domain with a phenylalanine residue resulted in a UBA1 domain that significantly increased the steady-state levels of the reporter substrate, although not to the same extent as the wild-type UBA2 domain (Fig. 4d). Turnover analysis showed that this single amino-acid substitution in the UBA1 domain indeed prolonged the half-life of the substrate (Fig. 4e). Our data suggest that the protective effect of the UBA2 domain is an intrinsic feature and identifies a well-conserved motif as a contributing factor.

Bottom Line:
We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains.Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains.Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

ABSTRACTThe ubiquitin receptors Rad23 and Dsk2 deliver polyubiquitylated substrates to the proteasome for destruction. The C-terminal ubiquitin-associated (UBA) domain of Rad23 functions as a cis-acting stabilization signal that protects this protein from proteasomal degradation. Here, we provide evidence that the C-terminal UBA domains guard ubiquitin receptors from destruction by preventing initiation of degradation at the proteasome. We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains. Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains. Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.